Comparisonofenamelremineralizationpotential afterapplicationoftitaniumtetrafluoride andcarbondioxidelaser

Comparisonofenamelremineralizationpotential afterapplicationoftitaniumtetrafluoride andcarbondioxidelaser Reza Fekrazad 1, Ahmad Najafi 2, Ramona Mahfar 3, Mahshid Namdari 4, Mohadese Azarsina 2 1: DDS, MSc, FLD, FICD; Associate professor of periodontology department, Dental Faculty- Laser Research Center in Medical Sciences, AJA University of Medical Sciences, Tehran, Iran. 2: DDS; Assistant Professor of Operative Dentistry, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3: Dental Student; Department of Operative Dentistry, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 4: PhD; Assistant Professor of Biostatistics Department, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Background and Aims: The aim was comparison of enamel remineralization after application of APF, TiF4 and CO2 laser alone or in combination. Materials and Methods: Enamel blocks were prepared from human third molars. The initial surface hardness was determined by Vicker's hardness tester. The samples underwent a demineralization regimen for 7 days to produce artificial initial caries. The hardness of enamel blocks with white spot lesions was measured, and the samples which had the mean hardness change of 65-90%, were selected, and randomly divided into 5 groups (N=15): G1: control; G2: APF 1.23%; G3: TiF4 4%; G4: TiF4 4% followed by CO2 laser (10.6 µm wavelength, 1 W peak power, 10 ms pulse duration, 500 ms repeat time, 0.2 mm beam spot size, 2 cm distance); G5: CO2 laser (same parameters) followed by TiF4 4%. Surface hardness recovery was measured after the treatments. Three samples in each group were observed under scanning electron microscope at x1,000 magnification. Data were analyzed by repeated measure ANOVA and Bonferrouni tests. Significance level was set at 0.05. Results: G2, G3, G4 indicated significant differences with control and G5 (p<0.05). Surface hardness in G5 was not significantly different from control (p=0.7) in enamel hardness test. There was not a significant difference between G2 & G3, G2 & G4, and G3 & G4 (p=1). The SEM results indicated globules of calcium fluoride on the surface in G2, and a smooth glaze-like surface layer in G3 and G4. In G5, some micro-cracks without any glaze-like layer were observed. Conclusions: APF, TiF4 and TiF4 before CO2 laser irradiation significantly increased the microhardness of initially demineralized enamel surfaces. CO2 laser irradiation before TiF4 application could not remineralize the white-spot lesions. Key words: Acidulated Phosphate Fluoride Laser Titanium Tetrafluoride Tooth Remineralization Addressee for Correspondence: Mohadese Azarsina: DDS, MS; Assistant Professor of Operative Dentistry Department, Dental School, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Department of Operative Dentistry, Dental School, Shahid Beheshti University of Medical Sciences, Daneshjo Blvd., Velenjak St., Tehran, Iran. Tel: +98 2129902305 Fax: +98 2188695955 Email: azarsina2012@yahoo.com Introduction Dental caries is a multifactorial infectious disease of the dental system with a dynamic process. It is known as the most common disease that affects dental tissues. It occurs due to the disruption of the balance between demineralization and remineralization of the teeth Received date: January 30th, 2017 Accepted date: April 3rd, 2017 2017 JMLL, Tokyo, Japan Laser Therapy 26.2: 113-119 113

available at www.jstage.jst.go.jp/browse/islsm occurring several times a day. 1) Incipient, non-cavitated enamel lesions have the potential to remineralize. Therefore, treatment modalities that rely on remineralization of the initially demineralized tooth, instead of conventional restoration strategies, are preferred methods for conservation of tooth structure. Fluoride is a known caries preventing agent. Various topical fluoride agents are used for caries prevention with different cariostatic potentials. Fluoride is also capable to expedite the tooth surface toward remineralization. 2) However, fluoride has only a partial cariostatic and remineralization potential. Acidulated phosphate fluoride (APF) of 1.23% is a known effective compound against tooth decay. The acidic nature of this compound causes increased fluoride uptake and more production of fluorohydroxyapatite during its chemical reaction with tooth mineral. 3) It can be studied as a suitable positive control for evaluation of new and especially acidic fluoride compounds. Titanium tetrafluoride (TiF4) has been studied since 1972 and has indicated promising preventive and therapeutic properties. It is reported as a stronger cariostatic agent compared to other fluoride compounds via formation of an acid-resistant coating composed of titanium dioxide or other organo-metallic complexes, and increased fluoride uptake due to the acidic nature of the compound. 4, 5) Carbon dioxide (CO2) laser was introduced in 1964. The wavelength of CO2 laser matches the absorption peak of water and hydroxyapatite in enamel (9-11 µm), so it gets absorbed well at the tooth surface. 6) The caries preventive effect of this laser can be related to reducing the permeability of the enamel by re-crystallization of enamel crystals into larger ones and removal of the enamel inter-rod substance. On the other hand, thermal changes by CO2 laser in the structure of phosphate acid and turning it into pyrophosphate may increase the acid resistance of enamel. 7, 8) Lasers are also reported as useful methods for promoting tooth remineralization especially when applied with fluoride. Although most of the studies have indicated better efficacy of the laser-fluoride combination therapy compared to single application of each one, 9-11) there is still no consensus on the most effective fluoride compound or the best treatment modality for the combination therapy. In addition, the effect of laser in combination with titanium tetrafluoride on remineralization of enamel was not studied before. Therefore, considering the importance of preventive techniques to reverse the caries process and remineralize the initially demineralized but not cavitated teeth, the aim of the present study was to compare the human enamel remineralization potential after application of TiF4 and CO2 laser alone or in combination. Materials and Methods: Erupted human third molars free from cracks, caries and any other faults were extracted and stored in 0.5% chloramine-t solution. Each tooth was sectioned with a cutting machine (MECATOME T201A, PRESI, Germany) into 5 enamel blocks (3 3 3 mm). The surface of each block was ground flat with watercooled carborundum discs (320, 600 grits; Buehler, Lake Bluff, IL, USA). The initial surface hardness (SH) of the samples was measured with a micro-hardness tester (HVS-1000, Laizhou Huayin Testing Ins,Taiwan). The hardness for each enamel block was reported as the mean of 3 indentations with 0/025 micron precision measurement on each sample, at 50 g force of the indenter applied for 10 seconds. Ninety blocks with Vicker's hardness of 233.51-301.81 kg/mm 2 were selected and then they were immersed in 50 ml of demineralizing solution (5 ml of NaN3, 1.5 ml of acetic acid, 2.9 g of NaCl, 0.12 g of CaCl2, 0.13 g of NaH2PO4 5 ml of NaF, ph = 4.8) for 7 days, in order to create artificial caries lesions as white spots. The artificial demineralization was checked and confirmed with a pathologic sample. At the second step, the hardness of the blocks with white spot lesions was measured the same way as the first step, and the percentage of surface hardness change was calculated for each sample [%SHC lesion= SH lesion SH primay/ SH primary] by a trained operator who was blind to the method and expected results. Seventy five blocks with mean %SHC of 65 90 were selected and randomly distributed into 5 groups (N = 15): Group 1; control: The samples were kept in artificial saliva without any treatment. Group 2; APF gel: The enamel blocks were treated with acidulated phosphate fluoride (APF) gel of 1.23% (Polio APF Fluid Effect, Imicryl, Turkey) for 4 minutes, and then the gel was washed out and air dried, as specified by the manufacturer. Group 3; TiF4: The enamel blocks were treated with 4% TiF4 gel for 1 minute, and then the gel was washed out and air dried. Group 4; TiF4 followed by CO2 laser: after applying the TiF4 gel for 1 minute on the blocks and washing and drying the samples, they were irradiated by CO2 laser with the following parameters; wavelength: 10.6 µm, peak power: 1 W, pulse duration: 10 ms, repeat 114 R Fekrazad et al

Table 1: Microhardness values of the samples in study groups by KgN/mm 2 Time T0 (Baseline) T1 (After demineralization) T2 (After treatment) Groups Mean SD Mean SD Mean SD Control 259.74 10.46 205.31 18.68 205.31 18.68 APF 265.25 16.34 211.29 19.42 276.02 24.68 TiF4 270.85 18.21 205.73 21.46 268.68 34.08 TiF4-Laser 266.99 20.94 215.49 23.01 264.05 27.97 Laser-TiF4 267.61 21.32 202.89 28.84 209.91 42.49 time: 500 ms, beam spot size: 2 mm, and distance: 2 cm. Group 5; CO2 laser followed by TiF4: The enamel blocks were irradiated by CO2 laser with parameters mentioned above and then the TiF4 gel was applied for 1 minute on the surface of the blocks and then washed out and air dried. After the treatments, the surface hardness recovery was measured as SHC = SH final SH lesion/ SH primary SH lesion. Three samples in each study group were observed by scanning electron microscopy (SEM) (VEGA2, TESCAN,USA) at 1000 magnification. The TiF4 gel with ph = 1.2 was prepared by dissolution of 4 g of TiF4 powder (Aldrich Chemical Company, Milwaukee, WI, USA) in 100 ml deionized water and addition of carboxymethyl cellulose immediately before the application. Data received by micro-hardness test was analyzed by repeated measure ANOVA and Bonferrouni tests and the significance level was defined at 0.05. Results: Surface micro-hardness values of samples at base line (t0), after demineralization (t1) and after treatments (t2) are listed in Table 1. Repeated measure ANOVA indicated significant differences of the groups 2, 3, 4 with control group and group 5 (p < 0.05). Surface hardness in group 5 was not significantly different from the control group (p = 0.7). Pairwise comparisons of the study groups by Bonferroni test are indicated in Table 2. SEM images illustrate the enamel surface structure of the study groups and the control group. We also prepared an SEM image for a normal, intact enamel sample to better understand the changes occurred on the surface after the treatments (Figs 1, 2). Discussion In the present study, acidulated phosphate fluoride was selected as a positive control that is known to be a potential remineralizing agent of initial caries. Hardness measurement is one of the useful methods to evaluate the mineral volume of tooth tissue which can be indicative of the possible demineralization or remineralization of the tissue when compared before and after a treatment modality, especially when other factors are kept stable. The results of micro-hardness evaluation indicated that APF and TiF4 were effective in remineralization of the white spot lesions. Laser treatment after TiF4 application was also effective in this regard, while laser Table 2: Pairwise comparison of the final microhardness values Groups Groups P-value Control APF TiF4 APF 0.0001 TiF4 0.0001 TiF4-Laser 0.0001 Laser-TiF4 0.7 TiF4 1 TiF4-Laser 1 Laser-TiF4 0.002 TiF4-Laser 1 Laser-TiF4 0.005 TiF4-Laser Laser-TiF4 0.014 Remineralization by TiF4 and CO2 laser 115

available at www.jstage.jst.go.jp/browse/islsm Fig. 1: SEM images of the samples in A: normal, intact enamel surface, B: demineralized enamel surface, C: enamel surface treated with APF 1.23% (globules of calcium fluoride on the surface), D: enamel surface treated with TiF4 gel (glaze-like layer was observed on the surface). Fig. 2: SEM images of the samples in A: enamel surface treated with TiF4 followed by CO2 laser (a glaze-like layer on the surface was visible), B: enamel surface treated with CO2 laser followed by TiF4 (some micro-cracks on the surface were exposed, without the glaze-like layer). 116 R Fekrazad et al

irradiation before the TiF4 therapy was not able to remineralize the initial caries lesions. The SEM observation of the study groups confirm the results of surface micro-hardness test. As observed in SEM images, APF created some globular structures of calcium fluoride on the tooth surface. The samples treated with TiF4 demonstrated a smooth and transparent glaze like layer on the enamel surface. The samples that were treated with TiF4 before CO2 laser exhibited a surface similar the ones that were treated only by TiF4. Laser treatment before TiF4 caused some micro-cracks on the enamel surface, without an obvious glaze-like layer related to TiF4. The efficacy of TiF4 is related to its titanium and fluoride ions. TiF4 protects the tooth by forming a protective barrier against noxious agents (mechanical protection), and increasing the fluoride uptake into the enamel surface (chemical protection). 5) In the aqueous environment of the oral cavity, TiF4 decomposes and changes into titanium dioxide (TiO2) which makes a resistant glaze-like layer on the tooth surface, and the acidic compound of HF. HF results in some surface porosities that increase the fluoride uptake on the tooth surface. 12) Like other fluoridated agents, the fluoride ion in TiF4 adheres to the calcium ions of the tooth surface and form CaF2 globules which replace hydroxyl ions released from the tooth structure during acidic challenges. The resultant fluoroapatite crystals are more acid resistant than hydroxyapatite. 5, 13, 14) The better fluoride uptake after application of TiF4 is related to the ability of its polyvalent metal ions to form organometallic complexes of fluoride that are strongly bonded to enamel hydroxyapatite molecules. 15) According to results of the present study, the effectiveness of TiF4 and TiF4 followed by laser was similar to the positive control group (APF), although they were all more effective in remineralization of the white spot lesions compared to the control group. These results are in accordance to the results of Alcantara et al that indicated the similar effectiveness of TiF4 and sodium fluoride (NaF) in remineralization of the initial caries lesions, and they were both more effective than the negative control group. 16) Comar et al. reported that TiF4 was as effective as sodium fluoride in prevention from caries lesion progression, while only TiF4 was able to remineralize the initial caries lesions. 17) The difference in the results between the two studies can be related to the extent of the caries lesion. It seems that in initial lesions all of the regular fluoride compounds can remineralize the enamel, but in more extensive caries, as seen in Comar s study, only TiF4 is able to remineralize the lesion. Another reason for the difference in results may be related to the acidic ph of APF used as the positive control material applied in the present research, compared to NaF used in Comar s study. Exterkate et al. reported more effectiveness of TiF4 compared to NaF in caries prevention. They reported that the number of application times and concentration of TiF4 were important in the results obtained. 15) In the present study, TiF4 was applied once during the study. Lasers are reported to have caries preventive effects on tooth enamel and dentin at specific settings. CO2 laser works at 9.3, 9.6, 10.3, and 10.6 µm wavelength, all of which match the absorption peak of hydroxyl, phosphate and carbonate groups of tooth mineral. 6) The caries preventive effect of this laser is related to the increase in surface temperature of the tooth by CO2 laser irradiation, which can result in obstruction of the exposed dentinal tubules, morphological and chemical changes in the tooth structure, a decrease in carbonate ions of enamel, and recrystallization of enamel hydroxyapatite molecules. 7, 8, 18-21) The combination of laser and fluoride therapy is reported more beneficial for caries prevention than laser irradiation or fluoride therapy alone. Laser irradiation of fluoridated tooth causes formation of a-tricalcium phosphate (TCP) and fluoroapatite. 22) Tetracalcium diphosphate monoxide is another compound formed on the tooth surface as the result of surface melting and carbonate removal, increasing the strength of tooth enamel. 23) It is suggested that if laser is applied after or through fluoride compound, the heat resulting from laser irradiation helps in fusion of the loosely-attached intermediate CaF2 crystals formed on the tooth surface 24) and also transform hydroxyapatite into fluoroapatite in presence of fluoride ion. 25) In the study of Poosti et al., CO2 laser before APF application, or the simultaneous treatment of teeth with CO2 laser and APF significantly increased remineralization of white-spot lesions and they were more effective than APF alone, or the control group. 26) The difference in their results with the present study may be related to the laser parameters. In their study, Poosti et al. irradiated 10.6 µm laser at 200 Hz, 10 mj energy, 10 W power, at 4 4 mm 2 surface area for 10 seconds with 25 mm distance from the samples. Decreased distance and smaller irradiation point of laser in the present study, may have increased the irradiance of the laser on enamel surface, with unfavorable mechanical alterations that were not beneficial for caries remineralization. In addition, laser irradiation might have Remineralization by TiF4 and CO2 laser 117

available at www.jstage.jst.go.jp/browse/islsm changed the organic volume of the enamel, so the mechanism of the function of TiF4, applied after laser irradiation, which depends on formation of organometallic compounds to form the protective glaze-like layer was impaired. In this situation, TiF4 with its low ph dissolved the mineral content of the enamel and exacerbated its mechanical properties. Anaraki et al. studied the effect of combination therapy of fluoride with CO2 and Er;Cr:YSGG lasers on enamel demineralization process. They observed better results for CO2 laser than Er;Cr:YSGG laser. The difference of this study with the present one is that the authors irradiated the laser beam on the fluoride-covered enamel surfaces, while in the present study fluoride gel was applied directly on the tooth surface before or after laser irradiation. 27) The results of the present study can be in agreement with this study in that we observed the positive effects of CO2 laser only on enamel samples which were modified in structure or chemistry, by the fluoride compound. Laser parameters play a key role in the caries preventive effects of various laser systems. Although higher irradiance of laser may better induce remineralization of the demineralized tooth or prevent the tooth from caries incidence, it may cause unfavorable macrostructural changes in enamel and dentin. Therefore, more studies are necessary to determine the best laser parameters with functional role in prevention and treatment of caries lesions, without any detrimental effects on the pulp or mechanical properties of the tooth. Within the limitations of the present study, it was concluded that APF, TiF4 and TiF4 before CO2 laser irradiation significantly increased the micro-hardness of initially demineralized enamel surfaces. These methods can be recommended as useful treatment protocols for white spot caries lesions. CO2 laser irradiation before TiF4 application could not remineralize the white-spot lesions. References: 1: Petersson GH (2003): Assessing caries risk--using the Cariogram model. Swedish Dental Journal, 158:1-65. 2: Clark MB, Slayton RL (2014): Fluoride use in caries prevention in the primary care setting. Pediatrics, 134(3):626-633. 3: Lee YE, Baek HJ, Choi YH, Jeong SH, Park YD, Song KB (2010): Comparison of remineralization effect of three topical fluoride regimens on enamel initial carious lesions. Journal of Dentistry, 38(2):166-171. 4: Magalhaes AC, Comar LP, Rios D, Delbem AC, Buzalaf MA (2008): Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralisation and remineralisation of bovine enamel in vitro. Journal of Dentistry, 36(2):158-162. 5: Wiegand A, Magalhaes AC, Attin T (2010): Is titanium tetrafluoride (TiF4) effective to prevent carious and erosive lesions? A review of the literature. Oral Health and Preventive Dentistry, 8(2):159-164. 6: Fried D, Ragadio J, Akrivou M, Featherstone JD, Murray MW, Dickenson KM (2001): Dental hard tissue modification and removal using sealed transverse excited atmospheric-pressure lasers operating at lambda = 9.6 and 10.6 microm. Journal of Biomedical Optics, 6(2):231-238. 7: de Melo JB, Hanashiro FS, Steagall W, Jr., Turbino ML, Nobre-dos-Santos M, Youssef MN, de Souza- Zaroni WC (2014): Effect of CO2 laser on root caries inhibition around composite restorations: an in vitro study. Lasers in Medical Science, 29(2):525-535. 8: Correa-Afonso AM, Bachmann L, Almeida CG, Corona SA, Borsatto MC (2012): FTIR and SEM analysis of CO2 laser irradiated human enamel. Archives of Oral Biology, 57(9):1153-1158. 9: Esteves-Oliveira M, Zezell DM, Ana PA, Yekta SS, Lampert F, Eduardo CP (2011): Dentine caries inhibition through CO(2) laser (10.6 µm) irradiation and fluoride application, in vitro. Archives of Oral Biology, 56(6):533-539. 10: Moslemi M, Fekrazad R, Tadayon N, Ghorbani M, Torabzadeh H, Shadkar MM (2009): Effects of ER,Cr:YSGG laser irradiation and fluoride treatment on acid resistance of the enamel. Pediatric Dentistry, 31(5):409-413. 11: Fekrazad R, Ebrahimpour L (2014): Evaluation of acquired acid resistance of enamel surrounding orthodontic brackets irradiated by laser and fluoride application. Lasers in Medical Science, 29(6):1793-1798. 12: Wiegand A, Waldheim E, Sener B, Magalhaes AC, Attin T (2009): Comparison of the effects of TiF4 and NaF solutions at ph 1.2 and 3.5 on enamel erosion in vitro. Caries Research, 43(4):269-277. 13: ten Cate JM, van Loveren C (1999): Fluoride mech- 118 R Fekrazad et al

anisms. Dental Clinics of North America, 43(4):713-742. 14: Moreno EC (1993): Role of Ca-P-F in caries prevention: chemical aspects. International Dental Journal, 43:71-80. 15: Exterkate RA, Ten Cate JM (2007): Effects of a new titanium fluoride derivative on enamel de- and remineralization. European Journal of Oral Sciences, 115(2):143-147. 16: Alcantara PC, Alexandria AK, Souza IP, Maia LC (2014): In situ effect of titanium tetrafluoride and sodium fluoride on artificially decayed human enamel. Brazillian Dental Journal, 25(1):28-32. 17: Comar LP, Wiegand A, Moron BM, Rios D, Buzalaf MA, Buchalla W, Magalhães AC (2012): In situ effect of sodium fluoride or titanium tetrafluoride varnish and solution on carious demineralization of enamel. European Journal of Oral Sciences, 120(4):342-348. 18: Takahashi K, Kimura Y, Matsumoto K (1998): Morphological and atomic analytical changes after CO2 laser irradiation emitted at 9.3 microns on human dental hard tissues. Journal of Clinical Laser Medicine and Surgery, 16(3):167-173. 19: Kato IT, Kohara EK, Sarkis JE, Wetter NU (2006): Effects of 960-nm diode laser irradiation on calcium solubility of dental enamel: an in vitro study. Photomedicine and Laser Surgery, 24(6):689-693. 20: Kanto Z, Featherstone JD, Fried D (1998): Caries prevention by CO2 laser treatment: dependency on the number of pulses used. Journal of American Dental Association, 129(5):585-591. 21: Chen CC, Huang ST (2009): The effects of lasers and fluoride on the acid resistance of decalcified human enamel. Photomedicine and Laser Surgery, 27(3):447-452. 22: Mathew A, Reddy NV, Sugumaran DK, Peter J, Shameer M, Dauravu LM (2013): Acquired acid resistance of human enamel treated with laser (Er:YAG laser and CO2 laser) and acidulated phosphate fluoride treatment: An in vitro atomic emission spectrometry analysis. Contemporary Clinical Dentistry, 4(2):170-175. 23: Souza-Gabriel AE, Colucci V, Turssi CP, Serra MC, Corona SA (2010): Microhardness and SEM after CO(2) laser irradiation or fluoride treatment in human and bovine enamel. Microscopy Research and Technique, 73(11):1030-1035. 24: Hossain MM, Hossain M, Kimura Y, Kinoshita J, Yamada Y, Matsumoto K (2002): Acquired acid resistance of enamel and dentin by CO2 laser irradiation with sodium fluoride solution. Journal of Clinical Laser and Medical Surgery, 20(2):77-82. 25: Tepper SA, Zehnder M, Pajarola GF, Schmidlin PR (2004): Increased fluoride uptake and acid resistance by CO2 laser-irradiation through topically applied fluoride on human enamel in vitro. Journal of Dentistry, 32(8):635-641. 26: Poosti M, Ahrari F, Moosavi H, Najjaran H (2014): The effect of fractional CO2 laser irradiation on remineralization of enamel white spot lesions. Lasers in Medical Sciences, 29(4):1349-1355. 27: Anaraki SN, Serajzadeh M, Fekrazad R (2012): Effects of laser-assisted fluoride therapy with a CO2 laser and Er, Cr:YSGG laser on enamel demineralization. Pediatric Dentistry, 34(4):e92-6. [Acknowledgements] The authors would like to thank the dental research center and the vice chancellor of research, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Remineralization by TiF4 and CO2 laser 119